This proposal concerns the structure, evolution and expression of mammalian tubulin genes. These genes encode the Alpha- and Beta-subunits of microtubules, structures that perform an essential role in many vital organelles--cilia, flagella, the mitotic spindle and the cytoskeleton--of all eucaryotic cells. The proposed approach will involve 1) characterization by DNA sequence analysis of human Beta-tubulin genes that encode distinct Beta-tubulin isotypes. 2)a) Synthesis of peptide haptens corresponding to small carboxyterminal regions that are unique to individual Beta-tubulin isotypes, b) use of these haptens in hapten/carrier conjugates for the generation of Beta-tubulin isotype-specific monoclonal antisera, c) application of these sera in immunofluorescence experiments to determine whether two or more Beta-tubulin isotypes can coassemble in a single microtubule. 3)a) Isolation of cDNA clones encoding mouse Alpha- and Beta-tubulin sequences from cDNA libraries constructed from whole embryo and adult polyA+ mRNA, b) generation of gene-specific 3'-untranslated region subcloned probes, c) use of these probes in in situ hybridization experiments using cryostat sections of staged animals to study the tissue-specific developmental regulation of individual tubulin genes. 4) Use of Beta-tubulin gene/BPV shuttle vectors for the identification of sequences within human Beta-tubulin genes that are critical in determining changes in the level of cytoplasmic Beta-tubulin mRNAs that occur in response to modulation of the size of intracellular monomer pools. 5)a) Construction of cloned probes encoding Tau and MAP sequences, b) use of these probes i) to determine the number and evolutionary conservation of the sequences encoding these proteins, ii) to assess the response of Tau and/or MAP mRNA levels to changes in the level of tubulin monomer, iii) to study the developmental regulation of these proteins by in situ hybridization.
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